PROJECT SUMARRY Targeting dysregulated phosphorylation signaling by kinase inhibitors is a proven strategy and a focus in the development of anti-cancer treatments. However, variability in patient responses and drug resistance limit efficacy and often lead to therapy failure. The underlying mechanisms that determine drug efficacy are multifaceted and include the drug target itself, as well as associated signaling pathways. For instance, mutation or amplification of the targeted kinase, functional compensation by related kinases, reprograming of kinase signaling, and alterations in phosphatase signaling networks that antagonize substrate phosphorylation or directly modulate kinase activity are common. Tremendous progress has been made in deciphering the cancer kinome and its response to anti-cancer treatment. These studies were enabled by an array of innovative technologies, including a chemical proteomics strategy that utilizes kinase inhibitors immobilized on beads and mass spectrometry (MS). The majority of protein dephosphorylation is carried out by phosphoprotein phosphatases (PPPs). The PPP family consists of nine catalytic subunits that bind to regulatory and scaffolding subunits and assemble into hundreds of multi-subunit enzymes and function as distinct, selective signaling entities. Excitingly, the role and regulation of PPPs in cellular signaling in normal and cancerous tissue is beginning to emerge. While kinome profiling provides global information on phosphorylation reactions, no such technology exists for phosphatases; thus, we lack knowledge of the understudied, but equally important, dephosphorylation reaction in cancer. To address this gap in capability, we have established a chemical proteomics strategy called ?PIB- MS? for efficient affinity-capture, identification, and quantification of all endogenously expressed PPPs and their associated proteins (?PPPome?) in a single mass spectrometry analysis from limited protein amounts of cell, tissue, and tumor lysate. In this application, we further develop and mature this technology and assess its performance to identify and quantify the PPPome in breast cancer cell lines, upon perturbation by drug treatment, and breast cancer PDX tumor models, and in primary human breast cancer tumors. We will assess the performance of this technology in an inter-laboratory comparison to achieve broad implementation. Statement of Potential Impact. PPPs have emerged as critical signaling entities in cancer. However, currently no approach exists for rapid, quantitative, and comprehensive assessment of the endogenous PPPome and its dynamic changes associated with cellular stress, short- and long-term treatment with anti-cancer drugs, or development of drug resistance. PIB-MS is a highly innovative technology that provides these capabilities for the analysis of cell lines, mouse models of disease, and primary human tumors. PIB-MS will accelerate and enhance the molecular analysis of cancer in basic, translational, and clinical research through rapid and quantitative analysis of PPP signaling, its alterations, and its effects on compensatory pathways before, during, and after anti-cancer treatment and upon development of resistance.